The present invention relates to fibrinolytically active plasminogen activators of the tissue type, DNA-sequences coding for same, pharmaceutical compositions containing same and processes for their production.
In particular, this invention relates to tissue-type plasminogen activators (t-PA) which have been modified in such a way that a; the uptake of the enzyme by the liver is reduced and b; the enzyme is essentially resistant to inactivation by plasma inhibitors. As a result the modified t-PA:s covered by this invention are characterized by a longer biological half-life than the t-PA preparations (native or recombinant) previously used.
Another aspect of this invention relates to the expression of t-PA, native or modified, in eucaryotic cells. More particularly, the invention relates to specific DNA sequences containing mRNA processing signals and which induce high production of recombinant proteins in heterologous cells.
Vascular disorders such as myocardial infarction, pulmonary embolism, stroke, deep vein thrombosis, periferal arterial thrombosis and other vascular thromboses are caused by partial or total occlusion of a blood vessel by blood clots. The clot which consists of a fibrin network can be dissolved by firinolytic enzymes. Plasmin is one such fibrinolytic enzyme which is present in the blood as an inactive proenzyme, plasminogen. Plasminogen activators convert plasminogen to plasmin, which in turn degrades the fibrin to soluble fragments. Thus, plasminogen activators can be use to induce thrombolysis.
The tissue plasminogen activator is regarded to be highly suitable for thromblytic treatments since it is a physiological compound with affinity for fibrin, and which activates plasminogen efficiently only in the presence of fibrin (Camiolo et al, Proc. Soc. Exp. Biol. Med., 138, pp. 277-280, 1971 and Ranby, M., Biochem. Biophys. Acta, 704, pp. 461-469, 1982). Thus, it is a clot selective fibrinolytic agent suitable for intravenous administration. Other plasminogen activators such as streptokinase, a bacterial protein, or urokinase, isolated from urine, activates plasminogen but are not clot selective. As a result circulating plasmin is generated which may cause a haemorrhagic potential because the circulating plasmin degrades clotting factors such as fibrinogen, factor VIII and factor V.
Clinical studies have demonstrated the thrombolytic effectiveness of t-PA for treatment of acute myocardial infarction. (The TIMI Study Group, N. Enql. J. Med., 312, p. 932, 1985 and Verstraete et al, Lancet, 1, pp. 842-847, 1985). However, due to the rapid clearance of t-PA by the liver (Korninger et al, Thromb. Haemostas., 46, pp. 658-661, 1981) high doses 50-90 mg had to be given as a continuous infusion in order to induce efficient thrombolysis. The biological half-life of t-PA in man is only a few minutes Tiefenbrunn et al, Circulation, 71, pp. 110-116, 1985), and only a small fraction of the activator will actually reach the clot. Another factor which further reduces the amount of t-PA available for clot lysis is the reaction with plasma inhibitors. It has been shown that t-PA forms complexes with a number of plasma protease inhibitors including the recently discovered plasminogen activator inhibitors of endothelial and placental type. (Rijken et al, J. Lab. Clin. Med., 101, pp. 285-294, 1983; Wiman et al, J. Biol. Chem., 259, pp. 3644-3647, 1984; and Lecander et al, British Journal of Haemathology, 57, pp. 407-412, 1984).
The modifications of t-PA according to the present invention solves both the problem of the short biological half-life due to the liver clearance and the sensitivity to inactivation by plasma inhibitors.
The DNA sequences containing the information for t-PA and derivatives thereof can be introduced into appropriate vectors for expression in eucaryotic cells. The fibrinolytic activity produced by the transiently transfected or stably transformed host cells may be measured by using standard assays for plasminogen activators. The eucaryotic expression vectors described herein may be constructed by techniques well known by those skilled in the art, using components such as replicons, enhancers, promoters etc from natural sources or chemically synthesized by conventional procedures.
Established cell lines, as well as normal diploid cells, are suitable as hosts. A large number of different cell lines are usable for expression of t-PA or derivatives thereof. For example, different hamster cell lines such as CHOdxe2x88x92, CHOK1 and BHK, monkey cell lines such as CV-1 and COS, mouse cell lines such as C127 and 3T3, as well as human cell lines may be used. Other hosts such as insect cells as well as transgenic organisms may also be used for the production of t-PA or t-PA derivatives.
After introduction into a suitable host cell, the t-PA coding DNA sequences may be contained and propagated either as stably integrated into the host cell genome or in extrachromosomal form.
The sequences comprising the t-PA gene is preferentially present in the cells in multiple copies. Different strategies for amplification of t-PA gene copy number may be exploited. For stable integration of the vector DNA into the host cells chromosomal DNA, and for the subsequent amplification of the integrated vector DNA, an amplifiable selectable gene is included in the t-PA expression vector. Chinese hamster ovary cells (CHO) are presently preferred together with the dihydrofolate reductase (DHFR) gene as an amplifiable selectable marker gene. (Kaufman et al, Mol. Cell. Biol., 7, pp. 1750-1759, 1985). 
Another amplification system is based on the use of papilloma virus DNA, especially bovine papilloma virus 1 (BPV). All or part of the virus genome is used to obtain stable transformation of mouse cells such as C127 or 3T3. The viral genome contains information for the maintenance of the vector DNA as a stable extrachromosomal element at a high copy number Sambrook et al, Embo J., 1, pp. 91-103, 1985).
In the eucaryotic host-vector systems discussed above, the expression of t-PA molecules or variants of the t-PA molecule is influenced by different upstream and downstream regulating DNA elements.
We have isolated and characterized a DNA fragment (KGH 11) from a human genomic library , which contains downstream processing signals such as polyadenylation signal etc from the human t-PA gene. The appropriate DNA fragment contains a part of the sequence in the last exon of the human t-PA gene. Since this segment also is represented in the cDNA it provides the possibility to use a unique restriction enzyme site in the overlapping region as a fusion site for ligation of the two elements (FIG. 3) For the eucaryotic expression systems analyzed, the production levels from this homologous construction were significantly higher than from expression vector constructions which are identical except for the processing signals downstream of the t-PA gene.
Recombinant DNA and other biotechnological techniques have been employed in order to obtain efficient production of t-PA for treatment of vascular diseases. Promising clinical studies were first performed with t-PA isolated from human melanoma cells. (Coken D, Circulation, 72, pp. 18-20, 1985).
Amino acid sequence analysis of the human melanoma t-PA (Wallxc3xa9n et al, Eur. J. Biochem., 132, pp. 681-686, 1983) provided the necessary information for the synthesis of DNA probes which were used for the isolation of, first a partial cDNA (Edlund et al, Proc. Natl. Acad. Sci. USA, 80, pp. 349-352, 1983), and subsequently a complete cDNA coding for the entire protein (FIG. 1).
Other examples where cDNA:s coding for t-PA have been isolated and where attempts have been made to produce t-PA in heterologous cells are referred to in European patent applications 93619 and 178105. See also reference (Pennica et al, Nature, 301, pp. 214-221, 1983).
Amino acid sequence determinations of various preparations of human t-PA have revealed differences in the N-terminal starting position. Due to differences in processing of the nascent molecule L,S, and U-forms of polypeptides are produced (Pohl et al, FEBS Lett., 168, pp. 657-663, 1985). The L-form is characterized by having glycine as the N-terminal residue. The N-terminal residue of the S-form and the U-form is serine and valine respectively. The numbering system for the amino acid sequence of t-PA used herein is based on the S-form where the N-terminal serine is numbered 1. As a consequence the L-form N-terminal glycine is at position xe2x88x923, and the U-form N-terminal valine is at position 4. It is understood that the tissue plasminogen activator modified in accordance with the present invention encompasses all such variant forms.
Different parts of the t-PA molecule show homologies with parts of other proteins. As disussed in (Patthy, L, Cell, 41, pp. 657-663, 1985) the native t-PA molecule consists of five structural domains which have been termed xe2x80x9cfinger domainxe2x80x9d (F), xe2x80x9cgrowth factor domainxe2x80x9d (G), xe2x80x9cKringle domainxe2x80x9d (K1 and K2 for Kringle 1 and Kringle 2) and xe2x80x9cprotease domainxe2x80x9d (P) respectively. Thus native t-PA can schematically be described by the formula:
F-G-K1-K2-P
The exon/intron junctions of the human t-PA gene have been determined (Ny et al, Proc. Natl. Acad. Sci. USA, 81, pp. 5355-5359, 1984), and the positions of these junctions can be used to define the boundries between the different domains in the amino acid sequence. Thus, the F domain consists of amino acid residues 4-50, the G domain consists of residues 51-87, the K1 domain consists of residues 88-176, and the K2 domain consists of residues 177-262. Following the K2 domain is a short connecting peptide consisting of the residues 263-274. The P domain is coded for by 5 exons and is contained within the amino acid sequence of residues 275-527 (Pohl et al, Biochemistry, 23, pp. 3701-3707, 1984). Human t-PA is synthesized as a single-chain 19 polypeptide but can be converted into a two-chain form where the two chains are connected through a disulfide bond. The heavy chain (residues 1-274) consists of the F, G, K1 and K2 domains together with a short connecting peptide, and the light chain (residues 275-527) consists of the P domain.
The fact that native, single-chain t-PA express activity with synthetic, low molecular weight, substrates and is inhibited by protease inhibitors (Ranby et al, Thromb. Res., 27, pp. 175-183, 1982) indicate that single-chain t-PA has significant enzymatic activity. In this respect, t-PA is different from other single-chain forms of serine proteases which have essentially no activity with synthetic substrates and does not react with inhibitors. It has been suggested (Wallxc3xa9n et al, Eur. J. Biochem., 132, pp. 681-686, 1983) that the enzymatic activity of the single-chain t-PA may be caused by the presence of a certain lysine residue (position 277 in the t-PA molecule). In all other serine proteases the corresponding position (i.e. position 2 after the activation cleavage site) is occupied by a small hydrofobic residue.
Attempts have been made to apply recombinant DNA techniques for the production of mutated forms of t-PA where the above mentioned lysine residue is exchanged for isoleucine (see International patent applicaton PCT/US85/01613). However, this modification is not expected to reduce the rapid uptake of t-PA by the liver since it has been shown that the rapid clearance is mediated by structures in the enzymatically inactive heavy chain of the molecule. (Rijken, D. C. and Emeis, J. J., Biochem. J., 238, pp. 643-646, 1986).
The main object of the present invention is to provide for fibrinolytically active plasminogen activators of the tissue type which have longer biological half-life in vivo. Another object of the invention is to provide activators which are less sensitive to inhibition by plasma inhibitors as compared with native human t-PA.
These and other objects of the invention which will be clear from the following disclosure are obtained by a fibrinolytically active plasminogen activator of the tissue type, wherein, in addition to the growth factor (G) domain, also the K1 domain has been deleted. Additionally the plasminogen activator of the invention has been modified in one or more of the following sites or region: the sites Of amino acid residues 177, 184, 277 and 448, and the F domain, if modified, being deleted in part or all of it.
It is preferred that the F domain, optionally, has been deleted and the 184 site has been modofied to prevent glycosylation thereat. It is particularly preferred that both sites 184 and 448 have been modified to provide glycosylation at said sites.
In this disclosure, when referring to modification of glycosylation sites 184 and 448, the modification is such that no glycosylation occurs. Thus, the site in question is modified so as to prevent N-glycosylation by modifying the N-glycosylation consensus sequence.
In a particularly preferred embodiment of the invention the F domain has been deleted altogether and the amino acid sites 184 and 448 have been modified to prevent glycosylation at said sites.
In such plasminogen activator it is preferred that the additional modification has been made at the site of amino acid residue 277, and such modification can be in the form of change to an amino acid residue which in its side chain does not exhibit a positive charge. An example of such modification is substituting a valine residue for the lysine residue at the 277 site.
In another preferred embodiment of the invention the additional modification has been made in the K1 domain, either as the only modification of the molecule in addition to the modification of the growth factor domain or in combination with the modification of the site of amino acid residue 277.
In yet another embodiment of the present invention modification of the molecule has been made at the site of amino acid residue 184, whereby N-glycosylation at said site, which occurs in normal t-PA, is no longer achievable. In a such modification at the 184 site the asparagine residue thereof can be replaced by a glutamine residue.
In addition to the said modifications at amino acid sites 184 and 277 it is also preferred to modify the K1 domain, optionally in combination with a modification to the F domain.
According to the present invention all such modifications to the different domains can be constituted by deletion or part of all of the respective domains.
The invention also covers a DNA-sequence comprising a nucleotide sequence encoding a fibrinolytically active plasminogen activator as described above. In addition, the invention comprises a replicable expression vector capable of expressing such a DNA-sequence. Furthermore, the invention includes host cells transformed with such replicable expression vector.
As indicated earlier in this disclosure the modified fibrinolytically active plasminogen activators according to the present invention displays a longer biological half-life as compared to native t-PA and is therefore particularly useful in pharmaceutical compositions and methods for the treatment of thrombotic diseases, such as vascular disorders.
The modified t-PA according to the present invention may be formulated using known methods for the manufacture of pharmaceutically useful compositions. Accordingly, the present invention also includes a pharmaceutical composition comprising a therapeutically effective amount of the modified t-PA in admixture with pharmaceutically acceptable carrier. The resulting compositions will provide an amount of modified t-PA effective in a patient to provide for treatment of thrombotic diseases, for example to dissolve blood clots.
Various dosage forms can be manufactured to enable administration of such pharmaceutical compositions. Thus, for example parenteral administration can be used for patients suffering from cardiovascular disorders. The dosage and frequence of administration will be selected according to the situation at hand. Because of the fact that the modified t-PA according to the present invention has been found to possess longer half-life than native t-PA the dosage can be significantly reduced compared to that presently used in therapy with prior art t-PA. Thus, quite generally, in the treatment of a patient for a thrombotic disorder there will be administered a daily dose of up to say about 1 mg/kg of body weight. Such administration can take place either by injection or by infusion.
Compositions for intravenous administration may take the form of solutions of the modified t-PA in an isotonic aqueous solution in sterile state. Such solution may contain a solubilizing agent to maintain the t-PA in solution.
According to another aspect of the invention there is provided a method of treating thrombotic disorders, which comprises administering to a patient suffering from such disorder an effective amount of the plasminogen activator according to the present invention.
In another aspect of this invention, these modified t-PA molecules which have an increased biological half-life compared with the native t-PA and have retained fibrin affinity, may be used for the in vivo localization of thrombi. The enzyme is preferably made inactive either by chemical modifications of the active site amino acid residues or by modifications of the DNA sequence coding for these residues. A number of well known methods exist for the chemical inactivation of serine proteases with agents such as diisopropylfluorophosphate (DFP), phenylmethylsulfonylfluoride (PMSF), N-p-tosyl-L-lysylchloromethane (TLCK) or peptide chloromethyl ketones such as H-D-Phe-Gly-ArgCH2Cl. Inactivations by genetic modifications may be performed by using site specific mutagenesis (as in example 2) and thereby change the DNA sequence coding for any of the active site residues. Preferably, the serine residue which corresponds to Ser-478 in the native, full sized, t-PA is changed to alanine.
The invention also covers DNA-sequences comprising a nucleotide sequence encoding a modified plasminogen activator according to the invention.
The invention also includes the preferred use of a DNA fragment containing the downstream mRNA processing signals provided from the human t-PA gene for the expression of human proteins in mammalian cells. The DNA fragment is characterized by the nucleotide sequence and restriction enzyme cleavage sites shown in FIG. 3.
Furthermore, the invention provides for a replicable expression vector capable of expressing, in a transformant host cell, such a DNA-sequence. In addition, the invention includes host cells transformed with such replicable expression vector.
According to yet another aspect of the invention there is provided a process for producing a modified plasminogen activator according to the invention, such process comprising:
a) preparing a replicable expression vector capable of expressing the DNA-sequence encoding such plasminogen activator;
b) transforming a host cell culture using the vector resulting from step a) to form redombinant host cells;
c) culturing said recombinant host cells under conditions permitting expression of the plasminogen activator encoding DNA-sequence to produce said plasminogen activator;
d) recovering the resulting plasminogen activator.
In such process eucaryotic host cells may be used.
By way of example, one compound of this invention differs from the native human t-PA by lacking the growth factor domain and the first Kringle domain. The amino acid residues from Pro-47 to Glu-175 of the native t-PA are deleted and Val-46 is directly followed by Gly-176. An other difference between this exemplary compound and the native t-PA molecule is that the N-glycosylation site in the second Kringle domain (present at Asn-184 in the native molecule) is made unavailable for glycosylation by the conversation of this asparagine residue to glutamine. The lysine residue at the position which corresponds to Lys-277 in the native t-PA molecule is changed to valine. The compound modified as above is denoted FK2(Gln)P(Val). In an other exemplary compound the residues from Cys-6 to Cys-173 of the native t-PA molecule are deleted, and here Ile-5 in the amino acid sequence is followed by Ser-174. The residues corresponding to Asn-184 and Lys-277 in the native molecule are modified in the same way as in the first compound. This second compound is denoted K2(Gln)P(Val). In addition, both FK2(Gln)P(Val) and K2(Gln)P(Val) are mutated to substitute the Asn residue at position 177 with Ser.
These exemplary compounds are schematically depicted in Table 1.
The numbers given for the residues refer to the native human t-PA sequence (see FIG. 1.)
The modifications of human t-PA according to the present invention is preferably a combination of modifications or a deletion of the growth factor domain, removal of at least one glycosylation site and modification of the second residue in the protease domain (Lys) into an amino acid residue which does not exhibit a positive charge in its side chain.
These fibrinolytically active modified t-PA molecules have longer biological half-life in the blood stream and are less sensitive to inactivation by complex formation with inhibitors than the native, unmodified, t-PA (natural or recombinant). Efficient thrombolysis may be obtained with comparatively lower doses of these mutant forms of t-PA than what is presently used for the unmodified t-PA.
While not wanting to be bound by theory we speculate that smaller molecules such as the modified t-PA:s may diffuse faster into the clot and thereby induce thrombolysis more efficiently than the unmodified full sized t-PA. Our results also indicate that these smaller t-PA molecules are expressed more efficiently by eucaryotic cells. Also potentially important for large scale production is that the single-chain form of the modified molecules will not react, or react more slowly than unmodified t-PA, with plasma inhibitors. This may increase the yield of fibrinolytically active molecules from tissue cultures, since in most cases the media have to be supplemented with serum which contain protease inhibitors. It has been reported that a significant part of the secreted unmodified t-PA is complexed to inhibitors derived from foetal calf serum (Schleuning W.-D., Abstract No. 82 of the VIII:th International Congress on Fibrinolysis, Vienna, 1986).
These modified fibrinolytic enzymes may be produced by means of recombinant DNA techniques. The DNA coding for the modified molecules may be constructed by digesting full length t-PA cDNA with suitable restriction enzymes, utilize techniques such as site directed mutagenesis and/or chemical synthesis of DNA fragments. These methods are well known by those who are ordinarily skilled in the art of recombinant DNA.
The DNA coding for the modified fibrinolytic molecules may be introduced into appropriate vectors for expression in eucaryotic or procaryotic cells.
Purification of the molecules may be conducted by procedures developed and known for native human t-PA with appropriate modifications. Proper purification procedures may be developed by persons ordinarily skilled in the art of protein purification.
By using a transcriptional unit which consists of an enhancer element as well as a promoter element upstream of the coding sequence for native or modified human t-PA fused to human t-PA downstream processing signals, high level expression were obtained in all eucaryotic cell systems analyzed, e.g. mouse cells such as C127, NIH 3T3, Swiss 3T3, hamster cells such as CHOdxe2x88x92, CHOK1, HAK, RS 1610, monkey cells such as CV-1, COS-1, COS-7. The expression levels were substantially higher by the use of homologous coding and downstream processing units, compared with using downstream processing signals from non-human eucaryotic genes or from genes of viral origin. The t-PA expression sequence is with respect to the sequence in the last exon and further downstream identical to the sequence for this region found in the human genome. This region is characterized by the nucleotide sequence and restriction enzyme cleavage sites indicated in FIG. 3.
This improved homologous unit is obtained by fusion of the element providing mRNA processing signals to a corresponding site in the last exon region of the cDNA.
Native and modified t-PA was injected as an intravenous bolus dose of 10-30 ug. From a cannula in the ear artery, frequent blood samples were collected and mixed with a 10% sodium citrate solution. After centrifugation the plasma samples were assayed for recombinant t-PA with an enzyme linked immunosorbent assay (ELISA) utilizing polyclonal antibodies raised against human melanoma t-PA.
Microorganisms, recombinant DNA molecules and the modified t-PA DNA coding sequences of this invention as well as starting materials useful in preparing them have been deposited in the culture collection of xe2x80x9cDeutsche Sammlung von Mikroorganismenxe2x80x9d, Grisebachstrasse 8, D-3400 Gxc3x96TTINGEN, Germany on Jun. 16, 1987 and have been identified there as:
A: E.coli JM83 (pKGE22) accession number: DMS 4142
B: E.coli HB 101 (pKGE81) accession number: DMS 4143
C: E.coli HB 101 (pKGE83) accession number: DSM 4144
D: E.coli HB 101 (pKGE105) accession number: DSM 4145
E: E.coli HB 101 (pKGE114) accession number: DSM 4146
The invention will be further understood with reference to the following illustrative embodiments, which are purely exemplary and should not be taken as limiting the true scope of the present invention as described in the claims.